pLJM1-FIRE-pHLy
(Plasmid #170775)

• Purpose: Ratiometric biosensor expressed with CMV promoter for probing lysosomal pH in mammalian cells
• Depositing Lab: Aimee Kao
• Publication: Chin et al ACS Sens. 2021 Jun 8. doi: 10.1021/acssensors.0c02318.

Backbone

• Vector backbone: pLJM1-EGFP
• Backbone manufacturer: Addgene Plasmid #19319
• Backbone size w/o insert (bp): 8083
• Total vector size (bp): 10093
• Modifications to backbone: EGFP replaced with FIRE-pHLy pH sensor (LAMP1 signal sequence-mTFP1-Linker1-humanLAMP1-Linker2-mCherry-stop codon)
• Vector type: Mammalian Expression, Lentiviral
• Selectable markers: Puromycin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: LAMP1
• Alt name: CD107a
• Species: H. sapiens (human)
• Insert Size (bp): 1167
• Mutation: 84 nucleotide Signal Sequence switched to the beginning of the construct
• GenBank ID: NM_005561.4 NM_005561.4
• Entrez Gene: LAMP1 (a.k.a. CD107a, LAMPA, LGP120)
• Promoter: CMV
• Tags / Fusion Proteins:
  • mTFP1 (N terminal on insert)
  • mCherry (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: CMV forward
• 3′ sequencing primer: hPGK promoter reverse

Resource Information

• Supplemental Documents:
  • pLJM1-FIRE-pHLy .seq
• A portion of this plasmid was derived from a plasmid made by: LAMP1 signal peptide and human LAMP1 were PCR amplified from LAMP1-mGFP (Addgene Plasmid #34831, kind gift from the Mark Von Zastrov lab, University of California, San Francisco, UCSF), mTFP1 amplified from mTFP1-pBAD (Addgene Plasmid #54553), mCherry amplified from pcDNA3.1-mCherry (Addgene Plasmid #128744). The DNA segments were PCR amplified (Phusion High-Fidelity PCR Master Mix, NEB, UK, #M0531) and fused with Gibson recombination cloning method (Gibson Assembly Master Mix, NEB, UK, #E2611) in pEGFP-N3 empty backbone. The linker sequences were incorporated into the primer sequences. The FIRE-pHLy expression cassette was cloned into lentiviral vectors with CMV promoter (pLJM1-EGFP; Addgene Plasmid #19319) by Epoch Life Science services (Sugar Land,Texas, USA).
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Chin et al. ACS Sens. 2021 Jun 8. doi: 10.1021/acssensors.0c02318.
Pubmed: https://pubmed.ncbi.nlm.nih.gov/34102054/
Article: https://pubs.acs.org/doi/10.1021/acssensors.0c02318